Amphotericin B derivatives

ABSTRACT

This disclosure describes two new water soluble derivatives of Amphotericin B which possess antifungal activity equal to or greater than the parent Amphotericin B.

United States Patent 191 McGahren et al.

[ 1 Oct. 21, 1975 AMPHOTERICIN B DERIVATIVES [75] Inventors: William James McGahren,

Demarest, N..l.; Martin Paul Kunstmann, Pearl River, NY.

[73] Assignee: American Cyanamid Company,

Stamford, Conn.

[22] Filed: Jan. 23, 1974 21 Appl. No.: 436,007

[52] U.S. Cl. 424/119 [51] Int. Cl. A61K 35/00 [58] Field of Search 424/1 19 [56] References Cited UNITED STATES PATENTS 2,908,611 l0/l959 Dutcher et al 424/1l9 Primary Examiner-Jerome D. Goldberg Attorney, Agent, or Firm-Edward A. Conroy, Jr.

[ ABSTRACT 1 Claim, 2 Drawing Figures AMPHOTERICIN B DERIVATIVES BRIEF SUMMARY OF THE INVENTION This invention relates to a novel group of water soluble derivatives of Amphotericin -B and, more particularly, is concerned with ether insoluble derivatives, one of which is an acidic derivative, and the other a neutral derivativeof Amphotericin B. The ether insoluble material is prepared by treating Amphotericin B with a stoichiometric amount of dry hydrogen chloride in methanol and recovering by precipitation with diethyl ether. This ether insoluble material is dissolved in water and the pH is adjusted to 3.0-4.0 for the acidic derivative, or to 6.2-6.7 for the neutral derivative, with a basic resin. Lyophilization of the clarified suspensions provides the acidic derivative or the neutral derivative, respectively.

DETAILED DESCRIPTION OF THE INVENTION Amphotericin B is a polyene macrolide antibiotic which is formed by fermentative biosynthesis during the cultivation under controlled conditions of Streptomyces nodosus. The preparation and properties of Amphotericin B are set forth in US. Pat. No. 2,908,611 which is hereby incorporated by reference. The structure of Amphotericin B is set forth in the Journal of the American Chemical Society, Vol. 93, No. 18, pp. 4560-4564 (1971) which is also incorporated by referenc.

The hydrochloride salt of Amphotericin B is described in US. Pat. No. 2,908,61 1. This salt is prepared by treating a solution of Amphotericin B in dimethylformamide with an equivalent amount of concentrated hydrochloric acid and recovering the product by precipitation with acetone. This salt is only slightly more soluble in water than the free base and the in vitro and in vivo antifungal activities of both are essentially the same. A slurry of the hydrochloride salt of Amphotericin B in water has a pH of 2.5 and upon adjustment of the pH to 7.0 virtually all of the Amphotericin B precipitates.

The hydrochloride salt of the methyl ester of Amphotericin B has been described by Mechlinski & Schaffner, J. Antibiotics 25, 259 (1972). However, the preparative method employed by these authors involves the use of diazomethane, which would render commercial preparation hazardous. Esterification of amino acids is. usually accomplished by suspending the amino acid in the appropriate alcohol and then bubbling dry hydrogen chloride through the suspension until solution is effected. The prior art, and in particular the reference cited immediately above, repute polyene macrolide antibiotics to be unstable under such conditions. Contrary to the teachings of the prior art, we have discovered that suspending Amphotericin B in dry methanol and treating this suspension (at -25C.) with a molar equivalent of hydrogen chloride gas dissolved in dry methanol followed by precipitation with diethyl ether provides a derivative of Amphotericin B which is very soluble in water. This ether insoluble material in water solution at a concentration of about 25 mg./ml. exhibits a pH of about 2.5. It is also not a methyl ester of Amphotericin B as indicated by spectral and thin-layer chromatographic tests.

Upon dissolving the ether insoluble material in water and adjusting the pH of the solution to 30-40 (at 025C.) with a basic resin, the dark red solution begins to form a yellow gel. This suspension is clarified by filtration of centrifugation and the clarified liquor is lyophilized to provide the acidic derivative of Amphotericin B as a bright yellow solid. Upon dissolving the ether insoluble material in water and adjusting the pH of the solution to 6.2-6.7 (at 0-25C.) with a basic resin, the yellow gel which forms at pH 3.0-4.0 redissolves to give a solution at pH 6.2-6.7. This suspension is clarified by filtration or centrifugation and the clarified liquor is lyophilized to provide the neutral derivative of Amphotericin B as a golden yellow amorphous solid. The basic resin which is employed for pH adjustment may be any water insoluble strong basic resin but preferably one having quaternary ammonium hydroxide groups. A typical basic resin of this kind is Amberlyst A26 which is available from the Rohm & Haas Company of Philadelphia, Pa. Amberlyst A26 is a polystyrene-polyamine type anion exchange resin having macroreticular properties.

The neutral derivative of Amphotericin B is soluble in water to the extent of 25-30 mg./ml. and the pH of its aqueous solution is in the neutral range. Although the acidic derivative of Amphotericin B contains between 1.3% and l.8% chlorine, the neutral derivative contains no chlorine. Both the acidic derivative and the neutral derivative are as stable as Amphotericin B and both derivatives exhibit antifungal activity equal to or greater than that of Amphotericin B. The ratio of the extinction coefficients at 385 my. and 407 my in the ultraviolet for Amphotericin B and the hydrochloride salt of the methyl ester of Amphotericin B is always in the range of 0.870 to 0.890. This may be expressed as follows:

E at 385 mp.

The same ratio for both the acidic derivative and the neutral derivative of Amphotericin B is in the range 0.910 0.950.

The in vitro activity of the novel derivatives of the present invention and the comparison with the in vitro activity of prior art compounds was determined by the well known agar diffusion technique against Candida albicans and Cryplococcus neoformans. The results of these tests are expressed as the extent of the zone of inhibition in millimeters at a specified concentration.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 Preparation of the Hydrochloride Salt of the Methyl Ester of Amphotericin B Diazomethane is generated in tetrahydrofuran from the reaction of 16.6 g. of N-methyl-N-nitroso-ptoluenesulfonamide) with 4 g. of KOH in the presence of a small quantity of ethanol. A yellow distillate of -85 ml. is collected. A 5 g. portion of Amphotericin B is stirred in 30 ml. of dimethylsulfoxide and cooled to 0C. The 80-85 ml. of diazomethane solution is added with stirring to the cooled slurry. The resulting dark red solution is run into 600 m1. of stirred diethyl ether. The yellow precipitate which forms is collected by centrifugation and dried yielding 5.0 g. of the methyl ester of Amphotericin B. This ester is suspended in 250 ml. of ice-cold stirred water and 0.1N HCL is added dropwise. The pH is monitored so that it is kept above 5.0. When 42 ml. of acid has been added, virtually all of the material is dissolved giving a straw-colored solu- EXAMPLE 3 Preparation of the Acidic Derivative of Amphotericin B tion. The reaction contents are centrifuged and the su- 5 Approximately 5 gm, of Amphotericin B are slurried pernatant solu ion i ly philiz y g -7 gof y lin 25 ml. of dry methanol at C. A solution of dry HCI 10W Solid Product E 1 cm. a! 407 u 1,400 gas in dry methanol is prepared which is approximately 3 1M in HCI. An aliquot of ml. of this acidic solution in mm actimy Zane of is added dropwise to the slurry with stirring so that esagainst Candida Concentration Inhibition sent1ally all the polyene 1s in solution. The dark red solution is stirred for a further minutes and the poly- HCI Sahofthe 200 183 ene is recovered by precipitation in diethyl ether fol- Methyl Ester of 100 16.0 lowed by centrifugation. The ether wet residue is dis- 3 1 3 3g 13:; solved in 200 ml of water and Amberlyst A26 [in the 100 17.3 15 hydroxyl form] is added with stirring until the pH 1s 50 about 4.0. Precipitation of the polyene begins to occur between pH 3.5 and 4.0. The resin is centrifuged off XAMPLE 2 and about one-third of the supernatant is freeze-dried Preparation of the Conventional Hydrochloride Salf of F yeld golden yellow f matefnal which is the acidic der1vat1ve of Amphotericin B w1th the fol- Amphotericm B lowing characteristics: A 4.7 g. portion of Amphotericin B is stirred for 1 hour in 120 ml. of dimethylformamide. A 0.42 ml. portion of concentrated hydrochloric acid is added dropwise resulting in a reddish solution having pH 3.7. This Elemental Analysis: C, 52 42; H, 7 77; N, 1.67; 01, 1.36. solution is filtered and 1,200 ml. of acetone is added to Rommm ig 'g d'methyl' the filtrate giving a light yellow suspension. The solid is recovered by filtration yielding 12 g. of solvent-wet 51mm 407 i (CHEOIU- material which is divided into two parts. One part is dried over P 0 in vacuo yielding 2.0 g. of dense yellow m... at 385 solid. The second part is stirred in 150 ml. of water to o a 407 0934- give a suspension from which the acetone is evaporated. Freeze drying yields 1.8 g. of yellow powder. Total chlorine 3.47%; calculated 3.84%. Specific optical rotation: [011 (C 0.270 dimethyl- A standard infrared absorption spectrum of the sulfoxide). E at 407 mp. 1,510 (CH OH). E acidic derivative of Amphotericin B prepared in a KBr W at 407 mp. 1,530 (Cl-l Ol-l) for Amphotericin B. disc is shown in FIG. 1 of the accompanying drawings.

Concentration Zone of Inhibition in vitro activity against Candida albicans mgJml. mm.

' 200 16.2 HCI Salt of Amphotericin B 100 15.3 15.5

200 17.0 Amphotericin B 16.2 50 15.7

In vitro activity against Crytococcus neot'ormans 200 17.5 HCl Salt of Amphotericin B 100 17.5 50 17.7

200 19.5 Amphotericin B 100 18.8 50 18.0

Concentration Zone of Inhibition In vitro activity against Candida albicans mgJml. mm.

4 200 18.8 Amphotericin B 100 17.5 50 17.2

200 22.9 Acidic Derivative of Amphotericin B 100 22.3 50 21.4

In vitro activity against Cryptococcus neoformans 200 22.9 Amphotericin B 100 22.3 50 21.4

200 26.5 Acidic Derivative of Amphotericin B 100 26.3 50 24.7

EXAMPLE 4 Elemental Analysis: Specific Rotation:

C, 56.04; H, 7.56; N,'1.51;C1, 1.73. ]n "=+250 (C, 0.258 in dimethylsulfoxide).

E m 407 mp.= 1490 (CHQOH E at 385 mu E M m 407 mu 0920' EXAMPLE 5 Preparation of the Neutral Derivative of Amphotericin E B A 10 gm. portion of Amphotericin B is slurried in ml. of dry methanol and 12 ml. of a solution which is 1.1N in dry HCl gas in methanol is added. This suspension is filtered and the polyene is recovered from the filtrate by precipitation in diethyl ether and centrifugation. The ether wet residue is dissolved in 500 ml. of water and the pH is adjusted to 6.7 with Amberlyst resin. The resin is removed by filtration and, following evaporation of the ether, the filtrate is freeze-dried yielding 8.0 gm. of product with the following characteristics: I

Elemental Analysis: Specific Rotation:

C, 56.71; H, 7.62; N, 1.46; CI, 0.0. [a],,'-'-" +225 (C. 0.331 in dimethylsulfoxide).

E,,,,, at 407 lTlfL= 1370 (cu ou).

E m at 385 mu M at 407 m, 0.926.

Concentration Zone of Inhibition ln vitro activity against Candida albicans mg./ml. mm.

200 18.7 Acidic Derivative of Amphotericin B 17.3 50 16.8

200 17.8 Amphotericin B 100 17.3 50 17.2

In vitro activity against Cryptococcus neoformans 200 19.5 Acidic Derivative of Amphotericin B 100 19.3 50 18.7

1 200 19.3 Amphotericin B 100 18.8 50 18.8

Concentration Zone of Inhibition In vitro activity against Candida albicans mg./m1. mm.

200 22.3 Neutral Derivative of Amphotericin B 100 21.0 50 19.8

200 18.5 Methyl ester HCl salt of Amphotericin B 100 17.5 50 17.0

200 18.2 Fungizonc 100 17.0 50 [6.8

In vitro activity against Cryptococcus neoformans 200 24.5 Neutral Derivative of Amphotericin B 100 24.3 50 22.3

200 22.2 Methyl estcr HCl salt of Amphotericin B 100 22.8 50 22.0

200 18.3 Fungizonc 100 19.0 50 18.7

Fungizonc is an approximately 1:1 mixture of Amphotericin B and sodium desoxycholate. Concentration of Amphotericin B in Fungilone is calculated to allow for presence of sodium desoxycholate diluent.

EXAMPLE 6 Stability Studies on Amphotericin B Derivatives Solutions of 10 mg./ml. in water of the following mato about 7.2 with 0.2M NaOH solution. The resulting yellow dispersion was centrifuged and the supernatant solution was freeze-dried to yield 0.85 gm. of yellow powder labelled R. THe slurry-residue was dissolved in tenals f made: dimethylsulfoxide and the polyene precipitated with 1. Funglzone acetone to yield upon drying 0.6 gm. of solid labelled 2. Amphoter1c1n B Methyl Ester Hydrochlonde S 3. Neutral Derivative of Amphotericin B The solutions were placed in capped amber bottles at ambient temperature. The solutions were sampled for bioassay by shaking the bottle and removing 0.2 ml. The 0.2 ml. was added to 0.8 ml. of dimethylsulfoxide I 1,1335 and diluted to 10 ml. with water. For UV testing, 0.1 Mmem" l E 111407 mp. ml. samples were taken, added to 0.9 ml. of dimethylsulfoxide and then diluted to 5 mcg./ml. with methanol. 5 8 8238 A tabulation of the stability results appear in the fol- R 0.85 0.948 lowing Table. S 060 Stability Studies on Aqueous Solutions of Amphotericin B Derivatives Compound Test Day 0 Day 4 Day 7 Day 14 Day 21 Day Day 49 Day 63 Fungizone In vitro C. albicans 18.2 16.3 17.0 16.3 16.7 16.8 16.8 15.0 In vitro C. neoformans 18.3 18.3 17.3 16.3 17.5 17.0 16.5 17.0 E U at 407 my. 1560 1600 1580 1460 1520 1 1340 Amphotericin B ln vitro C. albicans 17.8 18.0 17.0 14.7 14.3 14.0 13.7 12.0 Methyl Ester In vitro C. ncoformans 20.2 21.0 19.7 16.8 16.0 14.8 13.7 13.5 Hydrochloride E,,,,, at 407 mu 1380 800 450 240 105 95 Neutral Deriva- In vitro C. albicans 22.3 20.0 20.0 18.2 18.0 17.3 17.5 16.3 tive of Ampho In vitro C. neoformans 24.5 23.3 21.0 20.7 19.5 8.5 19.7 17.7 tericin B E m 407 my. 1460 1300 1310 1030 1040 960 1020 ln vitro results given as zones of inhibition in mm.

EXAMPLE 7 3 EXAMPLE 8 5 To Illustrate the Difference Between the Acidic Derivative of Amphotericin B and the Conventional Hydrochloride Salt of Amphotericin B With Regard to Water Solubility Upon Neutralization a. Neutralization of HCl salt of Amphotericin B: About 1.9 gm. of the HCl salt of Amphotericin B was stirred in 100 ml. of H 0 to give a dispersion exhibiting a pH of 2.5. The pH was adjusted upward to 7.0 using 0.2M NaOH solution. The resultant suspension was centrifuged. The supernatant yellow dispersion was freeze-dried to yield mg. of yellow solid labelled P. The residue from centrifugation was washed with acetone and diethyl ether and dried in vacuo to yield 1.3 gm. of yellow solid labelled O.

b. Neutralization of acidic derivative of Amphotericin B: Approximately 1.9 gm. of the acidic derivative of Amphotericin B was dissolved in 100 ml. of H 0 to give a solution at pH 3.0. The pH was adjusted upward In vivo Activity of Amphotericin B Derivatives of Candida albicans.

Treatment: Mice were treated within 1 hour after initiation of infection with 0.5 ml. of drug suspension in sterile distilled water or 0.2% aqueous agar, administered intraperitoneally. Tests were observed and results recorded for 21 days.

The results for Amphotericin B derivatives appear in the following tables.

TABLE 1 In vivo Activity Against Candida albicans Alive/Total Mice Tested 21 Days After lnfection treated controls TABLE 2 In vivo Activity Against Cryplococcus neoformans Alive/Total Mice Tested 2] Days After Infection lnlrapcritoncal Amphotericin B Amphotericin B Amphotcricin Dose mgJkg' Acidic Derivative Neutral Derivative B 200 l8/20 50 1 1/20 l2.5 20/20 l9/20 /20 3.1 /20 5/20 0/20 0.8 0/20 3/20 0.2 0/20 0/ l9 Infected nontrcatcd controls 0/] 10 w l i d. has the following ratio of extinction coefficients in l. A neutral derivative of Amphotericin B, a comthe ultraviolet:

pound which a. is water soluble to the extent of 25-30 mg./ml. and

. 1% is effective in inhibiting the growth of fungi; and m m". at 385 u its essentially pure amorphous form Em... at 407 u 9104x950; b. has an optical rotation [a] =+225 +240 (in dimeth lsulfoxide); c has following elemental analysis (percent): c, 25 c. has a characteristic infrared absorption spectrum 56.71; H 762; N 1.46; as shown in FIG. 2 of the drawings. 

1. A NUETRAL DERIVATIVE OF AMPHOTERICIN B, A COMPOUND WHICH A. IS WATER SOLUBLE TO THE EXTENT OF 25-30 MG./ML. AND IS EFFECTIVE IN INHIBITING THE GROWTH OF FUNGI, AND IN ITS ESSEN-TIALLY PURE AMORPHOUS FORM B. HAS AN OPTICAL ROTATION (A)D25* = +2225* - + 240* (IN DIMETHYSULFOXIDE), C. HAS THE FOLLOWING ELEMENTAL ANALYSIS (PERCENT): C, 56.71, H, 7.62, N, 1.46, D. HAS THE FOLLOWING RATIO OF EXTINCTION COEFFICIENTS IN THE ULTRAVIOLET: 